This MBRS SCORE SC2 proposal will close a considerable gap in our knowledge on how vacuole/lysosome size and morphology impact the ability of the organelle to perform its degradative functions. Defects in this organelle's structur and function are implicated in reduced clearance of cytotoxic proteins, which contributes to Alzheimer's disease. Lysosome function also affects tumor genesis, and lysosome abundance and diameter become abnormally regulated in highly-metastatic cell models. My goals are to elucidate the regulatory mechanisms that govern vacuole size and morphology, and how these characteristics impact vacuole function. This proposal applies novel methods - including three-dimensional image analysis, and computational modeling - to determine how organelle structure impacts function on a cell-by-cell basis. In Aim 1, the PI and his students will (A) determine how vacuole biogenesis and inheritance are coordinated during the cell cycle and (B) if there is feedback regulation of vacuole size using quantitative modeling and experimental verification. We will measure the rates of vacuole growth and inheritance from time-lapse microscopy data. These data will also inform the development of a quantitative model to test if feedback regulation between cell and vacuole growth is necessary to regulate vacuole size. This model will be verified in experiments testing whether vacuoles recover from size perturbations due to genetic or environmental perturbations. In Aim 2, we will determine how the size and morphology of a vacuole impact its function in receptor down regulation. In close consultation with my faculty mentor, Prof. David Drubin at the University of California, Berkeley, we will develop a fluorescence assay for the rates of endocytosis and degradation of a cell-surface receptor. We will apply this assay to probe the effect of vacuole morphology mutations on the kinetics of receptor down regulation, as well as determine the independent effects of vacuole volume and surface area on down regulation kinetics. The findings of this proposal will provide a foundation of knowledge of the progression of neurodegenerative disease and cancer with implications for diagnosis and treatment of these diseases. To carry out this research plan, the PI will involve graduate and undergraduate researchers, with an emphasis on recruiting and training students from under-represented backgrounds.